Cell-based samples may become for some applications the preferred choice of screening in drug discovery research, potentially overtaking more traditional approaches that include animal models. The samples may be used to detect specific cellular pathways of chemical compounds, therapeutic proteins, synthetic ribonucleic acid (siRNA) agents and other structures of interest. Insights from these samples could enable more efficient discovery of effective drugs compared to non-cell-based samples, thus saving time and costs as well as the need for future secondary screens.
Cell-based samples may be used during the early phase of the drug discovery process such as for cell-based ion channel expression systems (e.g. hERG k+) in association with automated patch clamp assays. Using cell-based samples enables high throughput safety testing, such as cardiotoxicity or hepatoxicity of chemicals. For example, the introduction of desoxyribonucleic acid (DNA), siRNA, or other substances into cells is a micromanipulation technology applied to develop and optimize various cellular systems, which enables cell systems either to more closely approximate in vivo testing or to become more competent or more specific for various in vitro applications.
Methods for microinjecting (e.g., transfecting) substances into cell-based samples such as for example, DNA, siRNA or monoclonal antibodies (mAbs) may be divided into three categories: a) chemical carriers; b) Viral vectors used by biologists to deliver genetic material inside a living cell by infection, and c) physical procedures that introduce material directly into the cells. All those three methods have advantages and disadvantages depending on the type of application.
Chemical carriers generally comprise positively-charged liposomes that are specialized in transfecting different cell types. However, chemical carriers have a relatively high transfection success only in certain cell types. In addition, liposomes can cause cytotoxicity and may induce a potent unexpected cell response. These limitations for chemical carriers prevent analysis of more biologically-relevant cell types, and also can significantly affect experimental outcomes and drastically interfere with the understanding of a gene's function.
Viral vectors' asset lies in their ability to achieve a very high percentage of successful transfection. However, to successfully perform transfection, excellent operator skills are required. In addition, the operator may have to undertake distinctive safety measures. Viral vectors also have the drawback of unwanted influences on the cell-based samples and that the size of injectable DNA is limited. In any case, working with infectious or potentially infectious particles, coupled with the possibility of raising undesirable immune response from an organism, counterbalances the high transfection rates, especially for the clinical research.
Physical procedures of transfection may again be subdivided into three different categories. A first category refers to Electroporation systems, which uses electrical pulses to open up the membranes of mammalian cells for the passage of genes. A second category refers to microinjection, and a third category refers to Gene gun devices. Gene gun devices insert samples of DNA or RNA more directly and ensure more localized delivery. However, employing physical procedures may cause a relatively low viability of cell-based samples. Electroporation for example may kill about 50% of the cell based samples.
Substances may also be inserted into cell-based samples using manual or semi-automated microinjection procedures, which are ways of introducing DNA and various compounds for new drugs only into the type of cells that are adherent in culture. Generally, microinjection allows the introduction of molecules into a defined cell population at a known concentration, whilst the timing of the experiment is stringently controllable; several types of reagents may be simultaneously introduced into cells (e.g., DNA constructs may be co-injected with a labelled dextran to mark the injected cells); reagents may be introduced such as, for example, antibodies, peptides, siRNAs, dyes, and chemical substances, whilst generally maintaining the viability of the cells. Microinjection procedures require searching for a cell under the microscope, catching the cell with a patch-clamp-like pipette, positioning the micropipette, puncturing the cell with a glass hollow-needle (apex only few microns) and injecting the liquid containing the transfection material into the cell. Performing these steps manually may take up to 10-15 min per cell. Therefore, performing these steps using manual or semi-automated procedures for may be tedious and time-consuming. In addition, only a limited number of cells may be involved, which may not permit the subsequent analysis of effects upon some biochemical parameters. The major limitation of the approach is the small amount of microinjected cellular material obtained. Therefore, performing microinjection as known in the art can not be used in association with cell-based high throughput screening (HTS).
A semiautomatic microinjection apparatuses is disclosed in the following URLs by “Multi Channel Systems”: http://www.multichannelsystems.com/products-ion.html, and http://www.multichannelsystems.com/fileadmin/user_upload/Manuals/Roboocyte_Manual_ClampAmp2.pdf, both of which were visited on Jan. 19, 2009. Fujitsu discloses a microinjection apparatus in the following URL http://www.computers.us.fujitsu.com/www/products_bioscience.shtml?products/bioscience/cellinjector, which was visited on Jan. 18, 2009.